Project Summary/Abstract: The immune system, by and large, plays a primary role to minimize and/or prevent infection, in general, and periodontal disease, in particular. We propose that immunoregulatory abnormalities contribute to the pathogenesis of and susceptibility to periodontal disease. The fundamental hypothesis of our studies is that periodontal pathogens produce immunotoxins that alter local and/or systemic host defense, thereby enhancing their own virulence and/or that of other opportunisitic organisms. In this regard, our investigations have demonstrated that Aggregatibacter actinomycetemcomitans produces the cytolethal distending toxin (Cdt) which is capable of impairing human T-, B-cell and oral epithelial cell function by inducing cell cycle arrest and apoptosis. To date our studies have focused on A. actinomycetemcomitans Cdt subunit function, events critical to toxin-cell interactions, fate of Cdt-treated-cells and mode of action by which Cdt induces G2 arrest and apoptosis. In particular, we have demonstrated that Cdt subunits, CdtA and CdtC, comprise the binding unit which involves recognition and binding of CdtC to cholesterol in the context of cholesterol rich microdomains. Moreover, we have established a new paradigm for the molecular mechanism by which the active subunit, CdtB, acts to induce toxicity. Specifically, we have demonstrated that CdtB functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase mimicking the tumor suppressors, PTEN (ptase and tensin homolog deleted on chromosome ten) or SHIP (src homology 2-containing inositol phosphatase), and is able to block the phosphatidylinositol 3-kinase (PI-3K)/PIP3/Akt signaling cascade. This is in contrast to the findings of other investigators who have proposed that CdtB functions as a DNase inducing DNA damage. We have not been able to confirm similar events in lymphoid cells (25) and most recently we have demonstrated that PI-3K signaling blockade is also critical to toxicity in nonlymphoid cells including oral keratinocyte cell line (OKF6) and HeLa cells. The objectives of this proposal are to extend our observations (1) to link the effect of CdtB-induced PI-3K signaling blockade to cell cycle arrest and in turn apoptosis, (2) to establish the events that are crucial to CdtB internalization via cholesterol-enriched microdomains which in turn lead to retrograde transport and (3) to utilize our findings relative to the structure-function of Cdt subunits, and their functional domains, to identify relevant immunogenic peptides that are capable of neutralizing the toxin and/or inducing a neutralizing immune response. It is anticipated that the proposed study will provide important insight into the pathogenesis of disease caused by Cdt-producing bacteria such as A. actinomycetemcomitans and contribute to developing a rationale approach on which therapeutic intervention may be developed to prevent or limit disease.